Parasites of plant-eating insects are natural pesticides. One such solitary parasite, Microplitis croceipes (a member of the wasp family Braconidae), causes inhibition of growth and permanent developmental arrest of the tobacco budworm, the cotton bollworm (also known as the corn earworm and tomato fruitworm) and the soybean pod worm. The larvae of these insects (Heliothis and Helicoverpa spp.) feed on tobacco, cotton, maize, sorghum, soybeans, sunflower and tomatoes, among other plants. The larvae cause economic losses of over $1 billion annually, primarily in the form of yield reduction and costs related to control. Fitt, 34 Ann. Rev. Entomol. 17 (1989).
In the past, the United States Department of Agriculture has funded programs designed to increase Microplitis croceipes populations in Southern states so as to naturally combat these pests in those areas. These efforts were successful in reducing pest populations. Use of such xe2x80x9cbiomanagementxe2x80x9d techniques has generally been replaced with molecular biology techniques, such as those which allowed engineering of plants which express Bacillus thuringiensis toxin.
Certain extra-embryonic cells (teratocytes) from M. croceipes have been shown to be involved in impairment of the growth, development and related physiological parameters of Heliothis and Helicoverpa larvae. Although teratocytes do not undergo cell division subsequent to their release into the hemocoel of the host, they do become polyploidal. Injection of one larval equivalent of teratocytes caused characteristic post-wandering, pre-pupation developmental arrest and eventual death associated with parasitization. Zhang and Dahlman, 12 Arch. Insect Biochem. Physiol. 51 (1989). Teratocytes collected from M. croceipes eggs hatched in vitro produced responses similar to those collected from parasitized Heliothis larvae. Zhang et al., 43 J. Insect Physiol. 577 (1997). Furthermore, it has been suggested that when teratocytes are cultured in vitro, they secrete a mixture of proteins (teratocyte secreted proteins or TSP) which, when injected into host larvae, produced responses similar to parasitization. Schepers et al., 44 J. Insect Physiol. 767 (1998).
In related studies, juvenile hormone esterase and ecdysone titers have been shown to be suppressed by teratocytes to a degree similar to those in parasitized larvae. Zhang, et al. 20, Arch. Insect Biochem. Physiol. 231 (1992). Reduced titers of host hemolymph proteins have been observed, particularly in older stages, with a major effect on the 74, 76 and 82 kD storage protein monomers. Zhang et al., 43 J. Insect Physiol. 577 (1997). Inhibition of storage protein synthesis in the fat body was theorized to be at the level of translation, primarily based on the finding that storage protein mRNA levels did not change, even though protein synthesis declined precipitously after treatment with teratocytes. Dong et al., 32 Arch. Insect Biochem. Physiol. 237 (1996).
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of these documents.
It is therefore an object of the present invention to provide isolated nucleic acid compounds useful to inhibit insect larval growth and development.
It is a further object to provide isolated amino acid compounds useful to inhibit larval growth and development.
It is yet another object to provide methods to produce isolated amino acid compounds useful to inhibit larval growth and development.
It is yet another object to provide methods to inhibit larval growth and development.
In all of the above embodiments, it is an object to provide methods to reduce crop damage using the compounds and methods herein.
It is an object of the invention to provide a natural pesticide using the compounds and methods herein.
It is an object of the invention to provide vectors, cells and molecular constructs comprising the compounds and methods herein.
It is an object of the invention to provide plants, including plant parts, seeds and embryos comprising the compounds and methods herein.
Definitions:
For the purposes of the present application, the following terms have the following meanings. All other terms have the meaning as generally recognized in the art.
xe2x80x9cAllelic variantxe2x80x9d is meant to refer to a gene that occurs at essentially the same locus (or loci) as the referent sequence, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants can also comprise alterations in the 5xe2x80x2 or 3xe2x80x2 untranslated regions of the gene (e.g., in regulatory control regions).
xe2x80x9cFragmentxe2x80x9d is meant to refer to any nucleic acid or polypeptide subset of the referent compound.
xe2x80x9cInducing agentxe2x80x9d means any compound or condition that causes inducement of an inducible promoter, including chemical compounds or environmental conditions, such as drought, wounds, light cycle, etc.
xe2x80x9cMaizexe2x80x9d and xe2x80x9ccornxe2x80x9d shall be interchangeable and mean all maize varieties.
xe2x80x9cProteinsxe2x80x9d means any compounds which comprise amino acids, including peptides, polypeptides, fusion proteins, etc.
xe2x80x9cTransformxe2x80x9d means delivery of nucleic acid into a cell, including delivery which results in genomic integration and delivery which results in transient localization within the cell membrane.
Moreover, for the purposes of the present invention, the term xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d entity refers to one or more of that entity; for example, xe2x80x9ca proteinxe2x80x9d or xe2x80x9ca nucleic acid moleculexe2x80x9d refers to one or more of those compounds or at least one compound. As such, the terms xe2x80x9caxe2x80x9d (or xe2x80x9canxe2x80x9d), xe2x80x9cone or morexe2x80x9d and xe2x80x9cat least onexe2x80x9d can be used interchangeably herein. It is also to be noted that the terms xe2x80x9ccomprisingxe2x80x9d, xe2x80x9cincludingxe2x80x9d, and xe2x80x9chavingxe2x80x9d can be used interchangeably. Furthermore, a compound xe2x80x9cselected from the group consisting ofxe2x80x9d refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural milieu. As such, xe2x80x9cisolatedxe2x80x9d and xe2x80x9cbiologically purexe2x80x9d do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis.
Table 1xe2x80x94Sequence listing of cDNAs, peptides and primers.
Table 2xe2x80x94Responses of Heliothis virescens larvae to feeding on transgenic plants expressing the TSP gene.
Table 3xe2x80x94Responses of Manduca sexta larvae to feeding on transgenic plants expressing the TSP gene.